系统xc-是一种氨基酸反转运蛋白,将L-胱氨酸导入细胞并输出细胞内L-谷氨酸,以1:1的比例。由于L-胱氨酸是谷胱甘肽合成的重要前体,系统xc-通过谷胱甘肽为基础的抗氧化应激支持肿瘤细胞生长,被认为是癌症治疗的潜在治疗靶点。系统xc-由两个子单元组成,轻链亚基SLC7A11(xCT)和重链亚基SLC3A2(也称为CD98hc或4F2hc),它们通过保守的二硫键连接。尽管最近另一个SLC7成员的结构,L型氨基酸转运蛋白1(LAT1)与CD98hc复合,为理解氨基酸转运机制提供了结构基础,xCT的详细分子机制尚不清楚。为了揭示分子机制,我们对xCT-CD98hc复合物进行了单颗粒分析.由于野生型xCT-CD98hc稳定性差,无法纯化至同质,我们将一致诱变方法应用于xCT。与野生型相比,共有突变的构建体显示出增加的稳定性,并使低温电子显微镜(cryo-EM)图能够通过单粒子分析以6.2µ分辨率获得。低温EM图显示出足够的电子密度来分配二级结构。在xCT结构中,哈希和arm域得到了很好的解决,而束域显示出一定的灵活性。CD98hc位于xCT跨膜结构域旁边。本研究提供了xCT的结构基础,我们基于共识的策略可以代表解决不稳定蛋白质结构的一个很好的选择。
System xc - is an amino acid antiporter that imports L-cystine into cells and exports intracellular L-glutamate, at a 1:1 ratio. As L-cystine is an essential precursor for glutathione synthesis, system xc - supports tumor cell growth through glutathione-based oxidative stress resistance and is considered as a potential therapeutic target for cancer treatment. System xc - consists of two subunits, the light chain subunit SLC7A11 (xCT) and the heavy chain subunit SLC3A2 (also known as CD98hc or 4F2hc), which are linked by a conserved disulfide bridge. Although the recent structures of another SLC7 member, L-type amino acid transporter 1 (LAT1) in complex with CD98hc, have provided the structural basis toward understanding the amino acid transport mechanism, the detailed molecular mechanism of xCT remains unknown. To revealthe molecular mechanism, we performed single-particle analyses of the xCT-CD98hc complex. As wild-type xCT-CD98hc displayed poor stability and could not be purified to homogeneity, we applied a
consensus mutagenesis approach to xCT. The
consensus mutated construct exhibited increased stability as compared to the wild-type, and enabled the cryoelectron microscopy (cryo-EM) map to be obtained at 6.2 Å resolution by single-particle analysis. The cryo-EM map revealed sufficient electron density to assign secondary structures. In the xCT structure, the hash and arm domains are well resolved, whereas the bundle domain shows some flexibility. CD98hc is positioned next to the xCT transmembrane domain. This study provides the structural basis of xCT, and our
consensus-based strategy could represent a good choice toward solving unstable protein structures.